Abstract

Cholesterol oxidation products, also named “oxysterols”, were first mentioned and studied in 1913 by I. Lifschütz while developing the worldwide famous products Eucerin® and Nivea® cream. He described oxysterols of non-enzymatic origin, being principally oxygenated at the sterol ring. In contrary enzymatically derived oxysterols were discovered 50 years later and appeared to be mainly side chain oxygenated sterols. However, a few oxysterols of both origins exist. Nowadays, it is known that oxysterols tightly regulate cholesterol homeostasis that plays a major role in human health. This regulation takes place by the mean of four different pathways. The first is the inhibition of the commonly activated sterol regulatory element binding protein (SREBP) pathway and the second is the activation of liver X receptors α and β (LXRα and LXRβ). The third action of oxysterols is the accelerated degradation of the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), a key enzyme of cholesterol synthesis. The final pathway triggered by oxysterols is the enhanced cholesterol esterification for cell storage. These regulatory pathways as well as other oxysterol mediated mechanisms that do not regulate cholesterol levels, appeared in the last years to be important for several human physiological processes. For example it was found that 25-hydroxycholesterol (25-OHC) possesses anti-viral functions. This illustrates that the physiological importance of oxysterols was underestimated and that their implications are still not completely unravelled. Oxysterol analysis, however, is tricky and hampered by several difficulties, such as the large excess of cholesterol, the low endogenous concentrations of oxysterols, the possible autoxidation of cholesterol and oxysterols, the cellular localisation of oxysterols in membranes, the possible modifications of oxysterols like esterifications, reductions, or sulphations and finally their chemical structures and properties making mass spectrometry (MS) analysis difficult and not very specific. In this work, two liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods were established for the analysis of oxysterols in human diseases. The first method was developed and implemented for the analysis of oxysterols of non-enzymatic origin in a clinical setting of Niemann-Pick type C (NP-C) diagnosis. In addition, a second method was optimised for the analysis of enzymatically-derived oxysterols in the plasma of patients with Inflammatory Bowel Disease (IBD), opening a new research domain. In the first part dealing with the analysis of NP-C-specific oxysterols, we were able to precisely, accurately, and robustly measure Cholestane-3β,5α,6β-triol (C-triol) and 7-Ketocholesterol (7-KC) following a Steglich esterification. We established reference ranges for NP-C diagnostics, which turned out to be neither age nor gender-dependent and we established the pre-analytical stability of the compounds. We found an important intra-individual variation for both analytes, but could show increased C-triol levels in all Niemann-Pick patients including Niemann Pick type A and B patients. Our results suggest that especially the C-triol is a biomarker for all three Niemann-Pick diseases. In the second part dealing with the oxysterol fingerprint in IBD, we were able to separate in one chromatographic run 24(S)-hydroxycholesterol (24(S)-OHC), 25-hydroxycholesterol (25-OHC), 27- hydroxycholesterol (27-OHC), 7α,24(S)-dihydroxycholesterol (7α,24(S)-OHC), 7α,25-dihydroxycholesterol (7α,25-OHC), 7α,27-dihydroxycholesterol (7α,27-OHC), 7β,25-dihydroxycholesterol (7β,25-OHC), and 7β,27- dihydroxycholesterol (7β,27-OHC). The most important finding of our investigation in IBD is that 27-OHC and eventually 25-OHC are reduced in plasma of IBD patients compared to healthy volunteers. Both oxysterols are agonists for the LXR receptors, which are implicated in inflammation and thus may possibly participate in inflammatory mechanisms in IBD. The analysis of mono-and dihydroxycholesterols as biomarkers for IBD is a novel approach which shows promise but more IBD samples are required to corroborate the observed differences in oxysterol levels between the different groups, meaning the control group, patients with active Crohn’s disease or in remission, and patients with active Ulcerative colitis or in remission.

Abstract

Cholesterol oxidation products, also named “oxysterols”, were first mentioned and studied in 1913 by I. Lifschütz while developing the worldwide famous products Eucerin® and Nivea® cream. He described oxysterols of non-enzymatic origin, being principally oxygenated at the sterol ring. In contrary enzymatically derived oxysterols were discovered 50 years later and appeared to be mainly side chain oxygenated sterols. However, a few oxysterols of both origins exist. Nowadays, it is known that oxysterols tightly regulate cholesterol homeostasis that plays a major role in human health. This regulation takes place by the mean of four different pathways. The first is the inhibition of the commonly activated sterol regulatory element binding protein (SREBP) pathway and the second is the activation of liver X receptors α and β (LXRα and LXRβ). The third action of oxysterols is the accelerated degradation of the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), a key enzyme of cholesterol synthesis. The final pathway triggered by oxysterols is the enhanced cholesterol esterification for cell storage. These regulatory pathways as well as other oxysterol mediated mechanisms that do not regulate cholesterol levels, appeared in the last years to be important for several human physiological processes. For example it was found that 25-hydroxycholesterol (25-OHC) possesses anti-viral functions. This illustrates that the physiological importance of oxysterols was underestimated and that their implications are still not completely unravelled. Oxysterol analysis, however, is tricky and hampered by several difficulties, such as the large excess of cholesterol, the low endogenous concentrations of oxysterols, the possible autoxidation of cholesterol and oxysterols, the cellular localisation of oxysterols in membranes, the possible modifications of oxysterols like esterifications, reductions, or sulphations and finally their chemical structures and properties making mass spectrometry (MS) analysis difficult and not very specific. In this work, two liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods were established for the analysis of oxysterols in human diseases. The first method was developed and implemented for the analysis of oxysterols of non-enzymatic origin in a clinical setting of Niemann-Pick type C (NP-C) diagnosis. In addition, a second method was optimised for the analysis of enzymatically-derived oxysterols in the plasma of patients with Inflammatory Bowel Disease (IBD), opening a new research domain. In the first part dealing with the analysis of NP-C-specific oxysterols, we were able to precisely, accurately, and robustly measure Cholestane-3β,5α,6β-triol (C-triol) and 7-Ketocholesterol (7-KC) following a Steglich esterification. We established reference ranges for NP-C diagnostics, which turned out to be neither age nor gender-dependent and we established the pre-analytical stability of the compounds. We found an important intra-individual variation for both analytes, but could show increased C-triol levels in all Niemann-Pick patients including Niemann Pick type A and B patients. Our results suggest that especially the C-triol is a biomarker for all three Niemann-Pick diseases. In the second part dealing with the oxysterol fingerprint in IBD, we were able to separate in one chromatographic run 24(S)-hydroxycholesterol (24(S)-OHC), 25-hydroxycholesterol (25-OHC), 27- hydroxycholesterol (27-OHC), 7α,24(S)-dihydroxycholesterol (7α,24(S)-OHC), 7α,25-dihydroxycholesterol (7α,25-OHC), 7α,27-dihydroxycholesterol (7α,27-OHC), 7β,25-dihydroxycholesterol (7β,25-OHC), and 7β,27- dihydroxycholesterol (7β,27-OHC). The most important finding of our investigation in IBD is that 27-OHC and eventually 25-OHC are reduced in plasma of IBD patients compared to healthy volunteers. Both oxysterols are agonists for the LXR receptors, which are implicated in inflammation and thus may possibly participate in inflammatory mechanisms in IBD. The analysis of mono-and dihydroxycholesterols as biomarkers for IBD is a novel approach which shows promise but more IBD samples are required to corroborate the observed differences in oxysterol levels between the different groups, meaning the control group, patients with active Crohn’s disease or in remission, and patients with active Ulcerative colitis or in remission.

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